**8. Mechanism of insulin secretion stimulated by branched-chain keto-acids**

Postprandial response by insulin secretion is also given by substances other than glucose. These substances, which induce the secretion of insulin, are termed secretagogues in general. One important type of secretagogues is branched-chain keto-acids (BCKAs), metabolites of branched-chain amino acids (BCAAs) (**Figure 2**). We found that the alternative to the NOX4-mediated redox signaling exists for some other insulin secretagogues, particularly for BCKAs [1]. For the redox signaling in this case, the mitochondrial redox signaling replaced that one originating from NOX4. Thus we demonstrated that H2O2 signaling originating from mitochondria is essentially required for insulin secretion stimulated by BCAAs metabolized onto BCKAs, such as 2-ketoisocaproate (KIC; also termed 2-oxoisocaproate, OIC; leucine metabolite), 2-ketoisovalerate (KIV; valine metabolite) and 2-ketomethylvalerate (KMV; isoleucine metabolite) [166, 167]. This mechanism was evidenced by the effects of mitochondrial-matrix-targeted antioxidant SkQ1. We observed that SkQ1 did not affect GSIS in INS-1E cells, but completely inhibited insulin secretion stimulated by KIC [1]. Thus the NOX4 source of H2O2 cannot be efficiently inhibited by SkQ1 located within the inner phospholipid leaflet of the inner mitochondrial membrane, whereas the redox signaling originating from mitochondrion must be blocked.

Metabolism of BCKAs begins in the mitochondrial matrix by the reaction of the BCKA dehydrogenase complex (BCKDH), since there is no branched-chain amino acid aminotransferase (BCAT) in the cell cytosol [168]. BCKDH forms isovaleryl-CoA, isobutyryl-Co and methyl-isobutyryl-CoA from KIC, KIV and KMV, respectively. This is followed by a series of reactions resembling β-oxidation of fatty acids. This series, as well as FA β-oxidation, elevates formation of superoxide in the mitochondrial matrix by several ways. The major way is due to the reoxidation of the BCKDH co-factor FADH2 by the electron-transfer flavoprotein (ETF), the one electron carrier. Two electrons from the two ETF molecules are accepted by the electron-transfer flavoprotein: ubiquinone oxidoreductase (ETF:QOR) [169].

#### **Figure 2.**

*The mechanism of branched chain keto acid-stimulated insulin secretion involves redox signaling of mitochondrial origin. 2-ketoisocaproate (KIC), 2-ketomethylvalerate (KMV) and 2-ketoisovalerate (KIV) resulting from leucine, isoleucine, and valine, respectively, due to the branched chain aminotransferase reaction in mitochondria (BCAT2), are metabolized by the branched chain ketoacid dehydrogenase (BCKDH) in mitochondria. A series of reactions, BCKA oxidation leads to the electron transfer from the co-factor FADH2 of BCKDH, via ETF towards the ETF:QOR reaction reducing Q to QH2. This effectively retards the competing reaction of the Complex I of the mitochondrial respiratory chain, leading to the superoxide formation. In the mitochondrial matrix, superoxide is transformed to H2O2 by MnSOD, whereas by CuZnSOD in the intermembrane space and cytosol. The elevated mitochondrial/cytosolic H2O2 substitutes the redox signal of NOX4 origin. Consequently, such redox signaling, together with elevated ATP, allows the sufficient depolarization of the plasma membrane.*

**45**

*Redox Signaling is Essential for Insulin Secretion DOI: http://dx.doi.org/10.5772/intechopen.94312*

ETF:QOR itself may produce superoxide.

meal or an experimental high-fat diet [174].

enhanced OXPHOS.

ETF:QOR reaction is coupled to ubiquinone (Q ) oxidation to ubiquinol (QH2). This effectively competes with the Complex I reaction of the respiratory chain, also providing Q oxidation to QH2 driven by NADH. The electron transfer within the Complex I is thus effectively retarded and this results in a higher superoxide formation. Superoxide is then most probably increasingly formed at the IQ site (i.e. at the proximity of the Q-binding site), similarly as due to the reverse electron transfer. Alternatively, the Complex I electron transfer is retarded upon the acetyl-CoA entry (propionyl-CoA entry for KIV; through methylmalonyl and Succinyl-CoA) into the Krebs cycle. Also, acetoacetate influences redox homeostasis, as one of the final products of leucine metabolism. After superoxide conversion to the elevated H2O2 in the mitochondrial matrix, the H2O2 is elevated in the cytosol and thus represents the mitochondrial retrograde redox signaling. Its target could be again KATP (or KATP and TRPM2) which would depolarize the plasma membrane due to this redox signaling ongoing in parallel with the elevated ATP due to concomitantly

The BCAA oxidation involves the following sequence of reactions: isovaleryl-CoA dehydrogenase (IVD), methylcrotonyl-CoA carboxylase (MCC), methylglutoconyl-CoA hydratase (MGCoAH) and 3-hydroxy-3-methylglutaryl-CoA lyase (HMGCoAL). The end-products are acetyl-CoA and acetoacetate. Similarly, as for pyruvate metabolism via PDH, the common end-product acetyl-CoA drives the Krebs cycle. This may also increase mitochondrial superoxide formation. Acetyl-CoA is linked to the above acyl-CoA dehydrogenase reaction by the reaction of the ETF:QOR, using ubiquinone (CoQ or Q ) to oxidize it to ubiquinol QH. Also, the

In summary, independently of the molecular mechanism, BCAA metabolism leads to the increased mitochondrial superoxide formation. After conversion to H2O2 by the matrix MnSOD and the intermembrane space CuZnSOD, the ongoing H2O2 efflux from mitochondria can be regarded as redox signaling. We have clearly demonstrated that the absence of such redox signaling, for example, in the presence of the mitochondrial matrix-targeted antioxidants SkQ1 leads to a blockage of insulin secretion, which is otherwise stimulated with BCKAs [1]. Likewise, the silencing

Fatty acids (FAs) appear in pancreatic islet capillaries either bound to albumin or being part of postprandial chylomicrons resulting from dietary fat lipids. FA pool of lipoproteins can be also considered. The dietary fat lipids are rich in triglycerides, which are cleaved locally in pancreatic islet capillaries by lipoprotein lipase secreted by β-cells. Resulting 2-monoacylglycerol (2MAG) and long chain FAs [170–173] stimulate each own two receptors GPR119 [157] and GPR40/FFA1, respectively. Therefore, fatty acid-stimulated insulin secretion (FASIS) could be defined as the net insulin secretion induced at the low glucose concentration, which itself does not stimulate insulin secretion. It is still controversial, whether such a net FASIS exists, since some previous reports observed that glucose should always be present for fatty acid to induce insulin secretion response. In contrast, the other reports described FASIS at 3 mM glucose, but not at zero glucose. Physiologically, postprandial responses should be due to all secretagogues resulting from major saccharide, fat and protein components. FASIS may dominate late responses upon feeding by fatty

Theoretically, FASIS must concern with the two components (**Figure 3**). The first one should depend on metabolism and the second one should rely on the stimulation

of BCKDH led to the inhibition of insulin secretion stimulated with BCKAs.

**9. Mechanism of fatty acid-stimulated insulin secretion**

*Redox Signaling is Essential for Insulin Secretion DOI: http://dx.doi.org/10.5772/intechopen.94312*

*Type 2 Diabetes - From Pathophysiology to Cyber Systems*

mitochondrion must be blocked.

keto-acids (BCKAs), metabolites of branched-chain amino acids (BCAAs) (**Figure 2**). We found that the alternative to the NOX4-mediated redox signaling exists for some other insulin secretagogues, particularly for BCKAs [1]. For the redox signaling in this case, the mitochondrial redox signaling replaced that one originating from NOX4. Thus we demonstrated that H2O2 signaling originating from mitochondria is essentially required for insulin secretion stimulated by BCAAs metabolized onto BCKAs, such as 2-ketoisocaproate (KIC; also termed 2-oxoisocaproate, OIC; leucine metabolite), 2-ketoisovalerate (KIV; valine metabolite) and 2-ketomethylvalerate (KMV; isoleucine metabolite) [166, 167]. This mechanism was evidenced by the effects of mitochondrial-matrix-targeted antioxidant SkQ1. We observed that SkQ1 did not affect GSIS in INS-1E cells, but completely inhibited insulin secretion stimulated by KIC [1]. Thus the NOX4 source of H2O2 cannot be efficiently inhibited by SkQ1 located within the inner phospholipid leaflet of the inner mitochondrial membrane, whereas the redox signaling originating from

Metabolism of BCKAs begins in the mitochondrial matrix by the reaction of the BCKA dehydrogenase complex (BCKDH), since there is no branched-chain amino acid aminotransferase (BCAT) in the cell cytosol [168]. BCKDH forms isovaleryl-CoA, isobutyryl-Co and methyl-isobutyryl-CoA from KIC, KIV and KMV,

respectively. This is followed by a series of reactions resembling β-oxidation of fatty acids. This series, as well as FA β-oxidation, elevates formation of superoxide in the mitochondrial matrix by several ways. The major way is due to the reoxidation of the BCKDH co-factor FADH2 by the electron-transfer flavoprotein (ETF), the one electron carrier. Two electrons from the two ETF molecules are accepted by the electron-transfer flavoprotein: ubiquinone oxidoreductase (ETF:QOR) [169].

*The mechanism of branched chain keto acid-stimulated insulin secretion involves redox signaling of mitochondrial origin. 2-ketoisocaproate (KIC), 2-ketomethylvalerate (KMV) and 2-ketoisovalerate (KIV) resulting from leucine, isoleucine, and valine, respectively, due to the branched chain aminotransferase reaction in mitochondria (BCAT2), are metabolized by the branched chain ketoacid dehydrogenase (BCKDH) in mitochondria. A series of reactions, BCKA oxidation leads to the electron transfer from the co-factor FADH2 of BCKDH, via ETF towards the ETF:QOR reaction reducing Q to QH2. This effectively retards the competing reaction of the Complex I of the mitochondrial respiratory chain, leading to the superoxide formation. In the mitochondrial matrix, superoxide is transformed to H2O2 by MnSOD, whereas by CuZnSOD in the intermembrane space and cytosol. The elevated mitochondrial/cytosolic H2O2 substitutes the redox signal of NOX4 origin. Consequently, such redox signaling, together with elevated ATP, allows the sufficient* 

**44**

*depolarization of the plasma membrane.*

**Figure 2.**

ETF:QOR reaction is coupled to ubiquinone (Q ) oxidation to ubiquinol (QH2). This effectively competes with the Complex I reaction of the respiratory chain, also providing Q oxidation to QH2 driven by NADH. The electron transfer within the Complex I is thus effectively retarded and this results in a higher superoxide formation. Superoxide is then most probably increasingly formed at the IQ site (i.e. at the proximity of the Q-binding site), similarly as due to the reverse electron transfer.

Alternatively, the Complex I electron transfer is retarded upon the acetyl-CoA entry (propionyl-CoA entry for KIV; through methylmalonyl and Succinyl-CoA) into the Krebs cycle. Also, acetoacetate influences redox homeostasis, as one of the final products of leucine metabolism. After superoxide conversion to the elevated H2O2 in the mitochondrial matrix, the H2O2 is elevated in the cytosol and thus represents the mitochondrial retrograde redox signaling. Its target could be again KATP (or KATP and TRPM2) which would depolarize the plasma membrane due to this redox signaling ongoing in parallel with the elevated ATP due to concomitantly enhanced OXPHOS.

The BCAA oxidation involves the following sequence of reactions: isovaleryl-CoA dehydrogenase (IVD), methylcrotonyl-CoA carboxylase (MCC), methylglutoconyl-CoA hydratase (MGCoAH) and 3-hydroxy-3-methylglutaryl-CoA lyase (HMGCoAL). The end-products are acetyl-CoA and acetoacetate. Similarly, as for pyruvate metabolism via PDH, the common end-product acetyl-CoA drives the Krebs cycle. This may also increase mitochondrial superoxide formation. Acetyl-CoA is linked to the above acyl-CoA dehydrogenase reaction by the reaction of the ETF:QOR, using ubiquinone (CoQ or Q ) to oxidize it to ubiquinol QH. Also, the ETF:QOR itself may produce superoxide.

In summary, independently of the molecular mechanism, BCAA metabolism leads to the increased mitochondrial superoxide formation. After conversion to H2O2 by the matrix MnSOD and the intermembrane space CuZnSOD, the ongoing H2O2 efflux from mitochondria can be regarded as redox signaling. We have clearly demonstrated that the absence of such redox signaling, for example, in the presence of the mitochondrial matrix-targeted antioxidants SkQ1 leads to a blockage of insulin secretion, which is otherwise stimulated with BCKAs [1]. Likewise, the silencing of BCKDH led to the inhibition of insulin secretion stimulated with BCKAs.

#### **9. Mechanism of fatty acid-stimulated insulin secretion**

Fatty acids (FAs) appear in pancreatic islet capillaries either bound to albumin or being part of postprandial chylomicrons resulting from dietary fat lipids. FA pool of lipoproteins can be also considered. The dietary fat lipids are rich in triglycerides, which are cleaved locally in pancreatic islet capillaries by lipoprotein lipase secreted by β-cells. Resulting 2-monoacylglycerol (2MAG) and long chain FAs [170–173] stimulate each own two receptors GPR119 [157] and GPR40/FFA1, respectively. Therefore, fatty acid-stimulated insulin secretion (FASIS) could be defined as the net insulin secretion induced at the low glucose concentration, which itself does not stimulate insulin secretion. It is still controversial, whether such a net FASIS exists, since some previous reports observed that glucose should always be present for fatty acid to induce insulin secretion response. In contrast, the other reports described FASIS at 3 mM glucose, but not at zero glucose. Physiologically, postprandial responses should be due to all secretagogues resulting from major saccharide, fat and protein components. FASIS may dominate late responses upon feeding by fatty meal or an experimental high-fat diet [174].

Theoretically, FASIS must concern with the two components (**Figure 3**). The first one should depend on metabolism and the second one should rely on the stimulation

**47**

with mitochondrial fatty acids.

evidence.

*Redox Signaling is Essential for Insulin Secretion DOI: http://dx.doi.org/10.5772/intechopen.94312*

The receptor component of FASIS may be at least partly KATP-independent and even CaL-independent, hence may partly proceed independently of high glucose. In other words, FASIS at low glucose is theoretically possible. The major pathway downstream of GPR40 relies on Gαq/11, which induces PLC-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate into DAG and IP3 [110, 111]. The latter would amplify the primary CaL-mediated Ca2+ signaling for the insulin release by mediating Ca2+ release from ER via the Ca2+ channel of the IP3 receptor [143]. However, this would happen provided that some basal CaL would be initiated by the metabolic component, i.e. due to partial fatty acid metabolism by β-oxidation followed by H2O2 plus ATP elevations. Also, PKC could be activated downstream of GPR40 as being the main effector of DAG. The PKC pathway could increase the extent of plasma membrane depolarization since it activates TRPM4 and TRPM5 [144]. Moreover, this may act at low glucose, again providing that the initial triggering is ensured by the metabolic component, and so that certain basal H2O2 plus ATP elevations exist, leading to the KATP closure. Also, another route downstream of GPR40 would involve the Gαq/11-PLC-TRPC-induced Ca2+ efflux from ER [176]. As mentioned above, the TRPC1-Orai1-STIM1 complex was demonstrated to act during GSIS, while contributing to Ca2+ oscillations [46, 83, 84]. The action of such a complex has yet to be

studied during experimental FASIS as well as its dependence on CaL.

**10. Redox relay as a hypothetical carrier for redox signaling**

It has been established that the content of glutathione (GSH) is rather low in pancreatic β-cells [177–180], in contrast to the content of thioredoxins and

Also, biased (promiscuous) pathways of GPR40, i.e., those involving GαS-cAMP initiation of information signaling may exist and contribute to a certain extent to FASIS. Both downstream pathways of GPR40-GαS-cAMP stimulation, i.e. the PKA and EPAC2 pathway, could target components of IGV interactions with the plasma membrane, hence being independent of CaL. These speculations await experimental

Our in vitro and in vivo experiments with mice (unpublished) demonstrated that approximately 2/3 of the GPR40 response (amplitude of insulin secretion) is given by the amplifying mechanism due to the mitochondrial phospholipase iPLA2γ/PNPLA8 [175]. This phospholipase cleaves both saturated and unsaturated FAs from the phospholipids of mitochondrial membranes. The cleaved free FAs subsequently diffuse up to the plasma membrane, where they activate GPR40. Moreover, the phospholipase iPLA2γ is directly activated by the elevated H2O2 in the mitochondrial matrix. The reader may remain that this is just the FA β-oxidation, which via the increased superoxide formation, due to the function of ETF:QOR and respiratory chain, produces H2O2, while the concomitant OXPHOS provides elevated ATP. As a result, the sufficient plasma membrane depolarization is enabled. The proof of co-existence of the GPR40 receptor component and metabolic component of FASIS is suggested by the experiments when FASIS in iPLA2γ knockout mice or in its isolated islets yielded only ~30% insulin secretion peak in the 1st phase when compared to wt mice (Holendová B., Jabůrek M, et al., unpublished). Incidentally, a similar portion remains when GW1100 antagonist of GPR40 was applied. These results show that in parallel with the GPR40 pathway, a 1/3 portion of FASIS still results from FA β-oxidation, having a similar mechanism as described for ketoacids. The abolished FASIS in the iPLA2γ knockout mice then supports the existence of such an acute mechanism in vivo, when GPR40 is supplied

#### **Figure 3.**

*(A) Traditional ("standard") view of FASIS compared with (B) FASIS with GPR40 receptor supplied by the mitochondrial fatty acids mechanism ("novel view"). The mechanism of FASIS at low glucose appears to be predominantly mediated by GPR40. Its ligands are free long-chain FAs. An excessive supply of GPR40 ligands and hence a substantial amplification of the GPR40 downstream response is given by the redox-activated mitochondrial phospholipase iPLA2*γ*/PNPLA8. The phospholipase iPLA2*γ *is directly activated by the elevated H2O2 in the mitochondrial matrix and cleaves both saturated and unsaturated FAs from the phospholipids of mitochondrial membranes. The cleaved free FAs diffuse up to the plasma membrane, where they activate GPR40.*

of the metabotropic receptor GPR40. In vivo, FASIS-GPR40 axis is paralleled by a portion of insulin secretion stimulated via another metabotropic receptor, GPR119, to which monoacylglycerol (MAG) binds as the second major component of triglycerides. The metabolic component undoubtedly involves fatty acid β-oxidation, providing both ATP from the elevated OXPHOS and H2O2 from the enhanced superoxide formation by the respiratory chain and ETF:QOR, similarly as for BCKAs [175]. This component is thus directly dependent on KATP, since it leads to its closure and to the canonical downstream events identical to those during GSIS.

#### *Redox Signaling is Essential for Insulin Secretion DOI: http://dx.doi.org/10.5772/intechopen.94312*

*Type 2 Diabetes - From Pathophysiology to Cyber Systems*

of the metabotropic receptor GPR40. In vivo, FASIS-GPR40 axis is paralleled by a portion of insulin secretion stimulated via another metabotropic receptor, GPR119, to which monoacylglycerol (MAG) binds as the second major component of triglycerides. The metabolic component undoubtedly involves fatty acid β-oxidation, providing both ATP from the elevated OXPHOS and H2O2 from the enhanced superoxide formation by the respiratory chain and ETF:QOR, similarly as for BCKAs [175]. This component is thus directly dependent on KATP, since it leads to its closure

*(A) Traditional ("standard") view of FASIS compared with (B) FASIS with GPR40 receptor supplied by the mitochondrial fatty acids mechanism ("novel view"). The mechanism of FASIS at low glucose appears to be predominantly mediated by GPR40. Its ligands are free long-chain FAs. An excessive supply of GPR40 ligands and hence a substantial amplification of the GPR40 downstream response is given by the redox-activated mitochondrial phospholipase iPLA2*γ*/PNPLA8. The phospholipase iPLA2*γ *is directly activated by the elevated H2O2 in the mitochondrial matrix and cleaves both saturated and unsaturated FAs from the phospholipids of mitochondrial membranes. The cleaved free FAs diffuse up to the plasma membrane, where they activate* 

and to the canonical downstream events identical to those during GSIS.

**46**

**Figure 3.**

*GPR40.*

The receptor component of FASIS may be at least partly KATP-independent and even CaL-independent, hence may partly proceed independently of high glucose. In other words, FASIS at low glucose is theoretically possible. The major pathway downstream of GPR40 relies on Gαq/11, which induces PLC-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate into DAG and IP3 [110, 111]. The latter would amplify the primary CaL-mediated Ca2+ signaling for the insulin release by mediating Ca2+ release from ER via the Ca2+ channel of the IP3 receptor [143]. However, this would happen provided that some basal CaL would be initiated by the metabolic component, i.e. due to partial fatty acid metabolism by β-oxidation followed by H2O2 plus ATP elevations. Also, PKC could be activated downstream of GPR40 as being the main effector of DAG. The PKC pathway could increase the extent of plasma membrane depolarization since it activates TRPM4 and TRPM5 [144]. Moreover, this may act at low glucose, again providing that the initial triggering is ensured by the metabolic component, and so that certain basal H2O2 plus ATP elevations exist, leading to the KATP closure. Also, another route downstream of GPR40 would involve the Gαq/11-PLC-TRPC-induced Ca2+ efflux from ER [176]. As mentioned above, the TRPC1-Orai1-STIM1 complex was demonstrated to act during GSIS, while contributing to Ca2+ oscillations [46, 83, 84]. The action of such a complex has yet to be studied during experimental FASIS as well as its dependence on CaL.

Also, biased (promiscuous) pathways of GPR40, i.e., those involving GαS-cAMP initiation of information signaling may exist and contribute to a certain extent to FASIS. Both downstream pathways of GPR40-GαS-cAMP stimulation, i.e. the PKA and EPAC2 pathway, could target components of IGV interactions with the plasma membrane, hence being independent of CaL. These speculations await experimental evidence.

Our in vitro and in vivo experiments with mice (unpublished) demonstrated that approximately 2/3 of the GPR40 response (amplitude of insulin secretion) is given by the amplifying mechanism due to the mitochondrial phospholipase iPLA2γ/PNPLA8 [175]. This phospholipase cleaves both saturated and unsaturated FAs from the phospholipids of mitochondrial membranes. The cleaved free FAs subsequently diffuse up to the plasma membrane, where they activate GPR40. Moreover, the phospholipase iPLA2γ is directly activated by the elevated H2O2 in the mitochondrial matrix. The reader may remain that this is just the FA β-oxidation, which via the increased superoxide formation, due to the function of ETF:QOR and respiratory chain, produces H2O2, while the concomitant OXPHOS provides elevated ATP. As a result, the sufficient plasma membrane depolarization is enabled. The proof of co-existence of the GPR40 receptor component and metabolic component of FASIS is suggested by the experiments when FASIS in iPLA2γ knockout mice or in its isolated islets yielded only ~30% insulin secretion peak in the 1st phase when compared to wt mice (Holendová B., Jabůrek M, et al., unpublished). Incidentally, a similar portion remains when GW1100 antagonist of GPR40 was applied. These results show that in parallel with the GPR40 pathway, a 1/3 portion of FASIS still results from FA β-oxidation, having a similar mechanism as described for ketoacids. The abolished FASIS in the iPLA2γ knockout mice then supports the existence of such an acute mechanism in vivo, when GPR40 is supplied with mitochondrial fatty acids.
